Inappropriate function of genes can be caused by errors introduced into the genetic code itself or by faulty epigenetic mechanisms deciding which genes can or cannot be expressed. Failure to produce proteins in the correct amounts or at all can disrupt essential metabolic, regulatory or signalling pathways resulting in the development of disease.
A disease such as cancer is caused by failure of checks and balances that control cell growth and proliferation. Improper levels of transcription and translation of certain genes results in unregulated cell growth. Certain specific genetic mutations have been identified as linked to several types of cancer, and, for some cancer types, this information has been converted into clinical tests. (Casey et al., Hum Mol Genet. 1993 November; 2(11); 1921-7). Genetic variability is also one of the best documented causes in the inconsistency of tumor responses. For instance, it has been documented that KRAS mutations cause resistance to epidermal growth factor receptor (EGFR)-targeted therapy (Rizzo, Cancer Treatment Reviews. 2010).
Studies have demonstrated that alterations in DNA methylation can also cause cancer. DNA methylation is a chemical modification of DNA performed by enzymes called methyltransferases, in which a methyl group (m) is added to certain cytosines (C) of DNA. This non-mutational (epigenetic) process (mC) is a critical factor in gene, expression regulation. In normal cells, methylation occurs predominantly in regions of DNA that have few CG base repeats, while CpG islands, regions that have long repeats of CG bases, remain non-methylated. Aberrant methylation of CpG islands may cause transcriptional inactivation or silencing of expression of certain genes in human cancers (Okino et al., Molecular carcinogenesis 2007 October; 46(10):839-46). Assessing the methylation status of genes may help predicting a person's prognosis to cancer. It has also been shown that aberrant DNA methylation may affect the sensitivity of cancers to antineoplastic agents by altering expression of genes critical to drug response. A well-known example in humans is promoter hypermethylation of O(6)-methylguanine-DNA methyltransferase (MGMT), which predicts favorable outcome for glioblastoma patients treated with the alkylating agent temozolomide (Hegi, ClinCancerRes, 2004; Stupp, Lancet, 2009).
Specific gene mutations and altered methylation patterns have also been linked to the development of neurological, neurodegenerative diseases and cardiovascular disorders. For example, Patients with Rett syndrome have neurodevelopmental defects associated with mutations in MeCP2, that binds to methylated DNA. Other mutations such as those in the Presenilin 1 (PSEN1) gene, seem to represent the most common cause of monogenic Alzheimer Disease (Borroni et al., Neurol Sci. 2011 Aug. 6.). Neurodegenerative disorders such as Alzheimer (Mastroeni D et al., PLoS ONE. 2009; 4(8):e6617) and psychiatric disorders such as schizophrenia (Costa E et al. Expert Rev Neurother. 2009; 9(1):87-98) and depression (Deutsch S I et al., Clin Neuropharmacol. 2008; 31(2): 104-119) appear to have disease-specific methylation patterns as well. Abnormally methylated genes (Mastronardi F G et al., J Neurosci Res. 2007; 85(9):2006-2016.) have also been linked to multiple sclerosis. Additionally, DNA methylation was also found to be linked to several cardiovascular-related biomarkers, including homocysteine (Ingrosso D et al., Lancet. 2003; 361:1693-1699.) and C-reactive protein.
Mutated genes or genes with altered methylation patterns involved in key pathways can affect disease progression and have the potential to influence drug resistance and clinical outcome following therapy. Knowledge on both molecular events may allow a clinician to predict more accurately how a disease is likely to respond to specific therapeutic treatments. Sequence specific amplification techniques have been developed for detection of sequence variations and alterations of wild-type locus. In standard PCR and sequencing reactions, information about mC and other covalent base modifications in genomic DNA is lost. As a consequence, indirect methods for DNA methylation analysis that after the genomic DNA in a methylation-dependent manner before amplification have been developed. Many methods that investigate DNA methylation use bisulfite treatment (Frommer, M., et al., Proc Natl Acad Sci USA 89 (1992) 1827-31). Bisulfite attaches itself to the C-6 of the cytosine ring. Subsequently, under alkaline conditions, the sulfonated cytosine is deaminated and desulfonated to uracil. The presence of a methyl group at the C-5 position prevents sulfonation and, therefore, methylcytosine remains the same. The bisulfite treated sequence can subsequently be assessed by a number of different methods such as bisulfite genomic sequencing (Grigg, G., et al., Bioesssays 16 (1994) 431-6; Grigg, G. W., DNA Seq 6 (1996) 189-98), nucleotide extension assays (MS-SNuPE), Pyrosequencing, Methylation Specific PCR (U.S. Pat. No. 5,786,146), MethyLight (WO 00/70090) and HeavyMethyl (WO 02/072880).
Bisulfite conversion is confronted with certain limitation such as incomplete conversion. The conditions necessary for complete conversion, such as long incubation times, elevated temperature, and high bisulfite concentration, can lead to the degradation of about 90% of the incubated DNA (Grunau C et al., 2001. Nucleic Acids Res. 29 (13): E65-5). Consequently, bisulfite conversion is applied only when required, for instance for methylation status determination.
Both mutation and methylation are involved in disease development and in patients' responses to particular drugs, and thus it is suitable to target multiple DNA alterations (Park et al., Int. 2006 J. Cancer: 120, 7-12). Since mutation and methylation detection methods operate according to two different principles, they do not lend themselves to be combined in a single assay. The method detecting mutations employs genomic DNA as a target, whereas the method detecting methylation requires bisulfite treatment. Accordingly, simultaneous mutation and methylation assessment currently requires the steps of splitting a nucleic acid sample in two parts and treating one part with bisulfite in order to allow methylation detection. This splitting is time consuming and has a negative effect on the efficiency of the process. Also, clinical samples are often small and splitting samples becomes often an issue.
Thus, there appears to be a need for improved diagnostic assays cancer using reliable and reproducible methods for determining DNA methylation and DNA mutation patterns simultaneously. This invention was made to address the foregoing need.